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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start Date of Experimental Work: October 28,1987 Completion Date of Experimental Work: December 14, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with some minor deviations from standard test guidelines and/or minor methodological deficiencies, which limit the reliability of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: The study was conducted in compliance with the Good Laboratory Practices (GLP) regulations of the U.S. Environmental Protection Agency (EPA), (40 CFR, Part 792) and according to the protocol and. standard operating procedures.
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical State :Liquid
Odour :Pungent
Specific Gravity :1.22 at 15.6°C
Solubility :See below for explanation
Storage Conditions :Room temperature
Safety Precautions :Avoid topical and respiratory contact

The test compound was provided by the sponsor. Portions were aliquoted into smaller containers prior to use. Due to difficulty in dissolving it in common vehicles such as saline or corn oil, the test material was administered to mice by oral gavage without use of a solvent.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Strain B6C3Fl mice were used in the study. Male and female mice were obtained from a reputable supplier. The males were dosed when they
were 13 weeks old while the females were dosed when they were 12-14 weeks old. Mice were quarantined at least one week and were group housed in cages with Easi-litter bedding. All animals were fed Agway Prolab R-MH 3000. They were given untreated Cambridge tap water supplied ad libitum. Animals were housed in a room with automatic light/dark cycle of approximately 12 hours each. Temperature of the environment was maintained at 72°F (± 10%). Each mouse was randomly assigned to code groups using computer generated random numbers, identified by ear punch, and individually caged following a quarantine period. Approximately eight hours prior to dosing feed was removed from the animals. One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in-this animal

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Due to difficulty in dissolving it in common vehicles such as saline or corn oil, the test material was administered to mice by oral gavage without use of a solvent
Details on exposure:
The induction of micronuclei in the bone marrow polychromatic erythrocyte cells as a result of chemical treatment is an established test for determining the genotoxic effect of chemicals. Micronuclei are cytoplasmic bodies with the appearance of small nuclei in the cell. These entities arise from chromosomal lagging at anaphase, from acentric chromosomal fragments, or from spindle fiber malformation during cell division. In mouse bone marrow, the cell population tested consists of erythroblasts undergoing their final mitosis before expulsion of the nucleus. The frequency of occurrence
of micronuclei in the polychromatic erythrocyte cells of treated animals provides an indicator of in vivo cytogenetic damage. Polychromatic erythrocytes or young red blood cells mature into normochromatic erythrocytes (NCEs) in the bone marrow. In normal untreated bone marrow cells, polychromatic erythrocytes and normochromatic erythrocytes are approximately in equal ratio. If a test agent is cytotoxic to nucleated ·cells, the proportion of polychromatic erythrocytes is reduced. The ratio of polychromatic erythrocytes to normochromatic erythrocyte is often used as an index of cytotoxicity of a test material.

A total of 80 mice, 40 males and 40 females, were used for the study.
Ten animals were used per test group. The animals were dosed once by oral gavage. Dosing volumes for each animal were determined from the weight of the animal, the specific gravity of the chemical, and the final dose desired. Dose volumes less than 0.1 ml were rounded to 0.1 ml. The target dose of the test chemical was 5 g/kg and the negative control was mineral oil at 5 ml/kg.

Time of Sacrifice (hours)
18 24 48
Test material 10 10 10
Mineral Oil 10 10 10
Cyclophosphamide(50mg/kg) 10


Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
once only
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
other: neat 5g/kg
No. of animals per sex per dose:
A total of 80 mice, 40 males and 40 females, were used for the study.
Ten animals were used per test group 5 x male 5x female
Control animals:
yes
Positive control(s):
Cyclophosphamide 50mg/kg

Examinations

Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
Bone Marow Collection
Following each exposure period (18, 24 and 48 hours), mice were killed by cervical dislocation and the femurs were removed and the ends cut. Bone marrow cells were collected by flushing the cavity with a phosphate buffered saline solution (pH 7.4).

Slide preparation
After collection of the bone marrow, the suspension was centrifuged and the supernatant was removed. Cells were resuspended in phosphate buffered saline solution. Slides were made by placing one drop of suspension on a clean slide and spreading the cells evenly with a second slide. At least two slides were made from each animal. Slides were labelled with the case number, chemical number, animal group number, animal identification number and an alphabet letter to designate replicate slides. Slides were than air dried at least 24 hours and fixed in 100% methanol for five minutes. Slides from mice treated with the test substance, and the negative control were coded, and the code was recorded in the original data notebook.

Staining
Acridine orange stain supplied by Sigma Chemical Co. (Lot No. l24F-3708) was made at a final concentration of 0.125mg/ml in sterile phosphate buffer saline solution (pH 7.4). Slides were stained a few minutes prior to analysis using a fluorescent microscope.
Evaluation criteria:
For each animal a minimum of 1000 polychromatic erythrocytes (PCEs) were counted for the presence of micronucleated PCEs. The frequency of micronucleated cells per animal was expressed as the number of micronucleated PCEs per 1000 PCEs' counted. The ratio of PCEs to normochromatic erythrocytes (NCEs) in 1000 total erythrocytes for each animal was recorded. The data were analyzed for statistical significance based on a binomial distribution, a level of significance of 0.05, and the tables of Kastenbaum and Bowman (Mutat Res ~ 527-549, 1970)
Statistics:
The data were analyzed for statistical significance based on a binomial distribution, a level of significance of 0.05, and the tables of Kastenbaum and Bowman (Mutat Res ~ 527-549, 1970)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in-this animal.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was tested for its genotoxicity using the mouse in vivo micronucleus screening assay. There was no significant increase in micronucleated PCEs in animals exposed to the test substance. Thus, the test material was negative in this in vivo assay.
Executive summary:

The test material was tested for its genotoxic activity using a mouse in vivo micronucleus screening assay. Male and female B6G3Fl mice were dosed once at 5 g/kg. The mice were killed at 18, 24 and 48 hours after dosing, and the bone marrow cells were collected. Ten animals, five males and five females were included in each group. At least one thousand polychromatic erythrocytes (PGEs) from each mouse were examined for micronuclei.

The negative control was mineral oil dosed at 5 ml/kg for 18, 24 and 48 hours, while the positive control was cyclophosphamide dosed at 50 mg/kg for 24 hours.

The mean micronucleated PGEs for negative control male mice were 2.6, 3.0 and 2.4 per 1000 PGEs for the three time periods. The average number of micronucleated PCEs for the positive control male mice was 14.5.

The mean micronuc1eated PCEs for the test chemical were 1.7, 4.3 and 3.6 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values.

The mean number of micronucleated PCEs for negative control female mice were 1.9, 2.1 and 2.9 per 1000 PCEs for the three time periods, while the positive control female mice had an average of 20.5. The frequencies of micronucleated PCEs in female mice treated with the test chemical were 2.9, 3.2 and 2.1 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values.

Based on these data, the test material which was tested at the maximum dose of 5 g/kg, did not induce a significant increase in micronuclei in bone marrow cells from either male or female B6G3Fl mice. Thus, the chemical was negative in this in vivo genetic toxicity test.